Detection of ethanol in Brazilian gasoline station attendants

AnalyticalMethods

PAPER

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article OnlineView Journal

Detection of eth

aGraduate Program in Pharmaceutical S

University of Rio Grande do Sul, 2752 Ipir

Alegre, Rio Grande do Sul, Brazil. E-m

33085437; Tel: +55-51-33085762bCenter for Drug and Alcohol Research – Hos

Federal University of Rio Grande do Sul –

Porto Alegre, Rio Grande do Sul, BrazilcDepartment of Statistics, Federal Univers

Gonçalves Avenue, 91509-200, Porto Alegre,

Cite this: DOI: 10.1039/c5ay00302d

Received 3rd February 2015Accepted 12th February 2015

DOI: 10.1039/c5ay00302d

www.rsc.org/methods

This journal is © The Royal Society of

anol in Brazilian gasoline stationattendants

Bruna Tassi Borille,*a Taıs Regina Fiorentin,a Bruna Claudia Coppe,a Eloisa Comiran,a

Ana Laura Bemvenuti Jacques,a Tanara Rosangela Vieira Sousa,b

Graciela Gema Pasa,b Flavio Pechansky,b Stela Maris de Jezus Castroc

and Renata Pereira Limbergera

In Brazil, gasoline station attendants are regularly exposed to the ethanol contained in fuel, as well as the one

used as the gasoline additive. This study aimed to assess the potential exposure of theseworkers to fuels, using

breathalyzers and oral fluid (OF) analysis by headspace gas chromatography/mass spectrometry (HS-GC/MS).

Attendants of 26 gasoline stations were invited to respond to a questionnaire covering themain features of the

study population and the profile of drinking and driving behavior, followed by a breath test and OF collection

with a Quantisal™ device. All OF samples were analyzed by HS-GC/MS. Ethanol was found in 100% of the OF

samples whereas 72.83% had concentrations above the quantification limit of the method (0.00125 g dL�1).

Regarding the breath tests, only one exhaled air sample (0.62%) showed a positive result (0.03 mg L�1). The

positive results in OF samples and negative results in exhaled air may be explained by the higher sensitivity

of OF analysis by HS-GC/MS, when compared to the breathalyzer.

1. Introduction

Road traffic injuries are a major cause of death globally and theleading cause of death for young people aged 15–29 years.1,2

According to the World Health Organization (WHO) it is esti-mated that more than one million people die annually in traffic-related accidents. Driving under the inuence (DUI) of alcoholor drugs is a major cause of traffic deaths.1,2

To reduce the number of traffic accidents related to DUI ofalcohol and drugs, the legal blood alcohol concentration (BAC)limit was introduced in Norway, in 1936.3 Currently in Norway,the legal BAC limit corresponds to about 0.02 g dL�1.4 Brazilintroduced the legislation limits corresponding to a BAC of 0.08g dL�1, in 1989, and in 2008 the “zero” limit was introducedwith suspension of driving privileges for a BAC above 0.02 gdL�1.3 Today, the legislation has recommended the limit ofbreath alcohol concentration (BrAC) to be 0.05 mg L�1 and“zero” tolerance for BAC. In cases that the BAC rate is higherthan 0.06 g dL�1 or for BrAC of 0.34 mg L�1, more severepunishments, including imprisonment, are imposed.5

ciences, College of Pharmacy, Federal

anga Avenue, Santana, 90610-000, Porto

ail: [email protected]; Fax: +55-51-

pital de Clinicas de Porto Alegre – UFRGS/

Rua Prof, Alvaro Alvim 400, 90420-020,

ity of Rio Grande do Sul, 9500 Bento

Rio Grande do Sul, Brazil

Chemistry 2015

The conversion values obtained in BrAC to the equivalentBAC are based on the principles of Henry's Law, which estab-lishes the relationship of BAC and BrAC as 1/2000, i.e., 2 liters ofexhaled air contains approximately the same amount of ethanolas in a milliliter of blood.6

A major concern regarding the use of breathalyzers is theirpotential inability to provide accurate results in the presence ofinterfering volatile organic components (e.g., cross-reacting),resulting in falsely increased ethanol reading.7,8 Breath, forinstance, is a rather complex mixture in which ethanol andacetaldehyde may be found. The origin of ethanol may greatlyvary, it may be inuenced by the environment, consideringacetaldehyde is a product of its metabolism.9 Acetone, methane,and isoprene are produced endogenously and, in highconcentrations, they may appear in the breath.10 Furthermore,ketone bodies may be produced by prolonged fasting, specicdiets, and in diabetic individuals.7,9,11 There may also be aresidual presence of ethanol in the mouth when recentlyconsumed, which can alter the blood/breath ratio, leading tofalsely elevated results in the reading equipment.7,11 Here, wehighlight workers directly exposed to ethanol during theworkday, as in the case of gasoline station attendants, who arethe focus of this research study.

It is crucial to conrm the positive results obtained from thebreathalyzer test through reliable and standardized laboratorytechniques, thus obtaining indisputable results. A recom-mended technique for conrmatory analysis of volatilesubstances in biological matrices is gas chromatography (GC)and headspace (HS),11,13 combined with mass spectrometry

Anal. Methods

Analytical Methods Paper

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article Online

(MS).13 Analysis by GC/MS allows the unequivocal identicationof analytes.12,13

Ethanol is the main component in alcoholic beverages. As achemical compound of low molecular weight (CH3CH2OH), it ispolar, highly soluble in water,14,15 it does not bind to plasmaproteins, and it is easily distributed to all tissues in the body,thus becoming a suitable compound for analysis in biologicalsamples such as blood, urine, oral uid, and sweat.15,16

In pharmacokinetic studies with ethanol, it was shown thatthere are large individual variations in its absorption, distri-bution, and elimination.17 This variability may occur due tofactors such as gender, age, race, consumption of alcoholicbeverages associated or not with food intake, exercise, alco-holism, and the use of some medications.18,19

Oral uid (OF) has been used worldwide for monitoring theconsumption of alcohol and drugs.20–27 The term “oral uid”refers to an organic matrix consisting of pure saliva secreted bysalivary glands, and a mixture of particles and uids found inthe oral cavity.26,28 This biological matrix presents severaladvantages over blood, which is usually employed for conr-matory analysis of ethanol concentration in drivers, such as thefact that the collection is noninvasive, easy to apply, fast, andpredictive, making it difficult for sample tampering by thegiver.16,24–27,29

Differences in substance concentrations in different matricesmay be explained by the fact that the transfer of substances fromblood to OF occurs by passive diffusion, primarily.29,33 Further-more, pH differences in the biological matrix, chemical prop-erties of substances, and substance–protein binding alsoinuence substance concentrations.21,30,31 Nevertheless, manyscientic studies have shown high correlations for ethanolbetween OF and blood,32,33,34,35 also ethanol concentration in OFreects positive correlations with exhaled air.36

Gubala and Zuba (2002) performed a controlled study ofethanol intake and measured its concentration in OF andplasma aer ingestion of alcohol, nding an average OF/plasmaratio of 1.08.20 Jones (1993) found a mean OF/plasma ratio of1.094, and this ratio remained the same during absorption,distribution, and elimination phases.37

Seeking to verify the correlation of ethanol concentration inexhaled air, OF, and urine, Bueno et al. (2014) observed a linearassociation between the concentrations of OF/exhaled air andOF/urine, using Pearson's correlation.36 The test showed that OFwas signicantly correlated with urine (0.93 – female; 0.91 –

male) and exhaled air (0.88 – female; 0.96 – male).36

In Brazil the fuels used and sold legally are: the ethanol fuel,gasoline, which is added in a percentage of 25% ethyl alcohol,and diesel, which has no alcohol in the composition. Thus thisstudy aimed to assess the potential interference of ethanol towhich gas station attendants are exposed, since these workersare subject to be approached in traffic aer the workday.

2. Materials and methods2.1 Chemicals, materials, and equipment

Ethanol and n-propanol were purchased from Tedia Company(Faireld, OH, USA). Quantisal™ OF collection devices, lters,

Anal. Methods

and preservative buffer solutions were donated by ImmunalysisCorporation (Pomona, CA, USA). HS vials and aluminum screwcaps with a PTFE/silicone septum were obtained from AgilentTechnologies (Agilent J&W Scientic, Folsom, CA, USA). GC/MSanalyses were performed on a GC 7890A coupled with a massdetector 5975C (Agilent Technologies, CA, USA), equipped withan automatic HS auto-sampler (CTC Analytics Combipal, Basel,Switzerland) and a ZB-BAC1 column (30 m � 0.32 mm � 1.80mm), from Zebron (Phenomenex). Alco-Sensor IV breathalyzerswith disposable mouthpieces were lent by the State TrafficDepartment of Rio Grande do Sul, Brazil.

2.2 Ethical issue

Ethical approval was granted by the ethical advisory board ofthe Federal University of Rio Grande do Sul, RS, Brazil, delib-eration number 23834513.4.0000.5347.

2.3 Sampling of exhaled air and oral uid

The sample size calculation for the comparison of two pairedgroups was performed using the null hypothesis, which postu-lates that the proportion of subjects with the presence ofethanol in exhaled air, detected by a breathalyzer is equal to theproportion of subjects with the presence of ethanol in OF,detected by HS-GC/MS. The calculation was performed at asignicance level of 5%, a correlation betweenmeasures of 0.79,and a magnitude of effect of 5%. For these parameters, 162sampling units had a statistical nominal power equal to at least80% to detect differences in proportions.

Paired exhaled air and OF samples were collected fromgasoline station attendants at the end of their shis. The studypopulation consisted of gasoline station attendants who, aerundergoing a questionnaire, had not drunk alcohol in the last 12hours. The questionnaire was applied with the aid of ODK Collectapp installed on tablets, followed by the application of thebreathalyzer. Oral uid samples were obtained usingQuantisal™OF collection devices, which contained a collection pad with anindicator that turns blue when 1 mL of OF has been collected.Aer collecting 1mL of OF the collection pad was transferred to aplastic transport tube with 3mL of buffering solution, with a nalspecimen volume of 4mL. A cylindrical lter was used to separateoral uid solution from the pad. All samples were transferred tothe laboratory using a Styrofoam box with monitored tempera-ture at approximately 5 �C, then they were frozen and stored nomore than 2 days at about – 10 �C until analysis.

2.4 Oral uid sample analysis

Oral uid samples were analyzed by the headspace gas chro-matography/mass spectrometry (HS-GC/MS) technique using apreviously published validated method.38 An aliquot of 1 mL ofOF sample with buffering solution was transferred to a HS vial,followed by the addition of 50 mL n-propanol 0.2 g dL�1 (internalstandard). The HS vials were sealed with PTFE/silicone septaand aluminum screw caps, and were placed into the vial rack ofthe automatic sampler, operating in the HS mode. The HS vialwas transported from the rack to the heater/stirrer and

This journal is © The Royal Society of Chemistry 2015

Paper Analytical Methods

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article Online

incubated for 7 min at 90 �C, with a stirring speed of 500 rpm.Finally, 1000 mL of gas phase was injected into GC/MS.

The injector was maintained at 200 �C and operated in splitmode 25/1. The oven temperature was programmed starting at40 �C for 3 min, with an increase of 5 �C min�1 to 70 �C, for 1min. The total run was 10 min. The post-run temperature wasmaintained at 200 �C for 3 min. Helium ultra pure was used asthe carrier gas at a ow rate of 1.4 mL min�1. Temperatures ofthe interface, ion source, and quadrupole were 220 �C, 230 �C,and 150 �C, respectively. The MS system was operated in elec-tron impact ionization mode at 70 eV, and in selected-ionmonitoring (SIM). The ions monitored were m/z 31, 45, 46 forethanol, and m/z 31, 59, 60 for n-propanol.

2.5 Data analysis

For data management and organization, Microso OfficeExcel™ soware was used and statistical analyses were per-formed using the SAS soware (v. 9.4).

Categorical variables were described by absolute frequencyand relative frequency, and compared between groups of genderusing Fisher's exact test. Quantitative variables were describedby mean and standard deviation and compared between groupsby the Mann–Whitney test.

3. Results3.1 Prole of gasoline station attendants

Paired samples of exhaled air and OF (n ¼ 162) were collectedfrom gasoline station attendants in 26 gasoline stations locatedin cities of Southern Brazil. One volunteer was excluded forfailing to satisfactorily provide oral uid and both men andwomen work, on average, 8 hours per day.

Table 1 summarizes overall features of our study population.On average, the population consisted of young (31.7 � 10.7years old), mostly male (70.99%) individuals, and both men andwomen were overweight considering their body mass index(BMI). Diabetes was reported by 4.3% of the participants, and45.8% of the total had a relative with the disease.

The behavior of gasoline station attendants about drinkingand driving, in this study, is shown in Table 2. Over half of thegasoline station attendants (63.3%) drive a car or a motorcycleevery day. In the last year, 128 gasoline station attendants (79%)drank alcoholic beverages in any quantity. From the totalsubjects who drank alcohol and drove (87), 29.9% had

Table 1 Main features of the study population

Total (n ¼ 162) Ma

Agea 31.7 � 10.7 32Heighta 1.70 � 0.09 1.7Weighta 76.6 � 14.6 79BMIa 26.6 � 4.90 26Diabetesb 7 (4.3%) 2 (Relatives with diabetesb,c 71 (45.8%) 27

a Mean � SD. b n (percentage). c (n ¼ 155). d Mann–Whitney test. e Fisher

This journal is © The Royal Society of Chemistry 2015

consumed alcoholic beverages immediately before driving.When asked if they had suffered traffic accidents, 58 subjectsanswered yes. Regarding the group of gasoline station atten-dants who have suffered accidents while driving (50), 94% (47)were driving at the time of the accident. From these 47 subjects,3 (6.38%) were DUI of alcohol at the time of the accident.

3.2 Exhaled air and oral uid

From 162 exhaled air samples, only one showed a positive result(0.03 mg L�1) for the breathalyzer test, while in OF samples, allsubjects, that is, 162 gasoline station attendants had ethanolconcentrations within the limits of detection of the method(0.0005 g dL�1).

From the 162 subjects with positive ethanol in the OFsample, 118 (72.83%) had ethanol concentrations above thelimit of quantication (0.00125 g dL�1) of the method, butbelow the lower limit of quantication (0.005 g dL�1) of thecalibration curve, except for one subject who showed an ethanolconcentration of 0.005 g dL�1 in the OF sample.

The exhaled air positive sample is from the same subject whoshowed an ethanol concentration of 0.005 g dL�1 in the OFsample. Since only one gasoline station attendant had a quan-titative level of ethanol in OF, from all gasoline stations, andonly one exhaled air sample showed positive results as well, theresults did not show variability, therefore it was not possible toconduct statistical tests for paired groups.

From 162 positives for ethanol in OF samples, whenanalyzed by age, the highest proportion of positives for ethanolin OF samples were from subjects aged 25–36 years (35.19%),followed by 18–25 years (33.95%) (Fig. 1).

Fig. 2 shows an example of a chromatogram obtained by theHS-GC/MS analysis for positive ethanol in OF samples, spikedwith an internal standard (n-propanol).

4. Discussion

The denition and enforcement of BAC limits of 0.05 g dL�1

may lead to signicant reduction in accidents related to alcoholconsumption worldwide. Since 2008 there has been an advancein laws on DUI, and currently, 66% of the world's populationapplies a BAC limit of 0.05 g dL�1 or less. This limit is justiedconsidering that above this concentration the risk of accidentssignicantly increases.2,3

le (n ¼ 115) Female (n ¼ 47) Signicance

.1 � 11.5 30.8 + 8.4 0.9882d

3 � 0.07 1.61 + 0.07 <0.0001d

.0 � 14.1 70.0 + 14.2 0.0006d

.2 � 4.29 27.5 + 6.09 0.2763d

28.6%) 5 (71.4%) 1.0000e

(38%) 44 (2%) 0.0326e

's exact test.

Anal. Methods

Fig. 1 Frequency of positive ethanol in OF (%) vs. age.

Table 2 Profile of drinking and driving behavior

Drive (n ¼ 162) Yes 103 (63.3%) Car 78 (75.7%)Motorcycle 25 (24.3%)

No 59 (36.4%)

In the last yearConsumption of any alcoholic beverage in any quantity (n ¼ 162)

Yes 128 (79%) 5 or more times a week 7 (5.5%)1 to 4 times a week 20 (15.6%)1 to 3 times a month 52 (40.6%)Less than one time per month 49 (32.3%)

No 34 (21%)

Dosesa of alcohol ingested in a normal day of consumption (n ¼ 128)1 to 5 doses 85 (66.4%)6 to 12 doses 31 (24.2%)>13 doses 12 (9.4%)

Drives aer work (n ¼ 103)Always 54 (52.4%)Almost always 14 (13.6%)Almost never 15 (14.6%)Never 20 (19.4%)

Consumed alcohol immediately before driving (n ¼ 87)Yes 26 (29.9%)No 61 (70.1%)

Has suffered a traffic accident (n ¼ 162)Yes 58 (35.8%)No 104 (64.2%)

Drives and was the driver at the time of traffic accident (n ¼ 50)Yes 47 (94%)No 3 (6%)

Drives, was the driver at the time of traffic accident, and was under the inuence of alcohol at the time of the accident (n ¼ 47)Yes 3 (6.38%)No 44 (93.62%)

a Dose: beer – 350 mL; wine – 90 mL; distillates – 50 mL.

Anal. Methods This journal is © The Royal Society of Chemistry 2015

Analytical Methods Paper

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article Online

Fig. 2 Chromatogram obtained by HS-GC/MS analysis of ethanol (1.691 min) in OF samples, spiked with an internal standard (n-propanol) (2.679min).

Paper Analytical Methods

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article Online

Oral uid proved to be a great biological matrix to be appliedin on-site collection approaches. The ndings through theapplication of OF in this study corroborates with the previouslymentioned advantages of this matrix, such as easy and quickcollection, the lack of constraint, the low potential fortampering by the subjects, and the lack of need for specializedprofessionals.

Although themost widely used technique for conrmation ofblood alcohol concentration in forensic laboratories is currentlyheadspace gas chromatography coupled with a ame ionizationdetector (HS-GC/FID), it allows the occurrence of false positiveswith alcohols and other volatile compounds, such as methanoland other solvent results. Thus, the use of HS-GC/MS may beconsidered the “gold standard” for conrmation of unques-tionable results.

With the results described, it may be inferred that OF anal-ysis by HS-GC/MS shows more positive results than breathaly-zers for ethanol analysis, when in very low concentrations, thatis, it seems to be more sensitive. While only one breathalyzertest showed positive ethanol results (0.62%), 100% (162subjects) of OF analysis by HS-GC/MS showed positive ethanolresults. These positive samples, 72.83% (118 samples) wereabove the quantication limit of the method, however, belowthe lower quantication limit of its calibration curve, and wereconsidered semiquantitative samples.

Although one sample was tested positive for the breatha-lyzer, the test reading was below the limit recommended formaximum permissible error, generating uncertainty about thetest, thus demonstrating the importance of conducting conr-matory analysis for the breathalyzer test, and the need for aconrmatory sample to be collected in the nearest possibleperiod from testing. Therefore, positive results in OF samples

This journal is © The Royal Society of Chemistry 2015

and negative results in exhaled air samples may be explained bythe fact that the method of analyzing OF by HS-GC/MS is moresensitive than by the breathalyzer based on electrochemical cellsensors.

Exposure to ethanol in fuels is probably responsible for100% of positive results in OF samples. Despite the lowconcentrations found in the OF of gasoline station attendants,the ethanol concentration, in this matrix, depends on theethanol concentration in the blood. However, in some trafficlegislations the limit allowed for ethanol in the blood of driversis equal to zero (e.g., in Brazil) so that any level of ethanoldetected may ultimately generate legal complications for thedriver.

An advantage of OF compared to blood is that OF can becollected at the location of the approach, immediately aer thebreathalyzer analysis. Rather for blood, it is required to movethe subjects to the laboratory, and depending on the delay, it ispossible that either the period of detection passes or a verydistinct concentration is found over the ethanol concentrationin the subjects when they were approached.

Due to the inability of breathalyzers to provide accurateresults in the presence of interfering volatile organiccompounds,7,8 such as ketone bodies produced by prolongedfasting, specic diets, and in diabetic subjects,7,8,11,39 the pop-ulation was stratied and assessed. Although 4.3% gasolinestation attendants have been reported to have diabetes, and45.8% reported that a close relative has the disease, neverthe-less no interference was observed in this study.

Although all population worked, on average, 8 hours per day,the frequency of positive ethanol in OF was higher among 26–35year old subjects, which is the same age group of the populationmajorly involved in traffic accidents, as demonstrated in several

Anal. Methods

Analytical Methods Paper

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article Online

studies, including in Brazil.4,40–44 According to the World HealthOrganization, traffic accidents are the second cause of deathamong persons aged 15 to 29 years.2

Because women have more fat and less body water per kg ofbody weight than men, they eliminate alcohol at a faster rate.Men and women generally have similar alcohol elimination rateswhen results are expressed as grams per hour. It is possible that agiven oral dose of alcohol may produce a higher BAC in womenthan inmen due to rst-passmetabolism.19,45 Although describedin the literature, differences in elimination rate between gasolinestation attendants were not observed since the exposure time forboth genders was the same and all individuals, both male andfemale, showed positive results for ethanol in OF samples.

5. Conclusions

In the present study we analysed the presence of ethanol ingasoline station attendants using breathalyzers, while qualita-tively and quantitatively analyzing OF by HS-GC/MS.

The results presented here demonstrate that breathalyzersare less effective when compared to OF analysis by HS-GC/MS,showing the importance of a conrmatory analysis for thebreathalyzer test. Although it was not possible to quantify all OFsamples, the breathalyzer test showed negative results while theOF samples by HS-GC/MS showed positive results.

As shown in several studies, this work proved that OFsamples are highly promising samples for forensic analysis,particularly for analyses where the collection of the biologicalmatrix is required at the site of approach.

Despite the results presented here showing an occupationalproblem among these workers, the aim of this study has non-occupational focus. The objective was to evaluate whether exposureof workers during working hours could interfere in application ofthe Brazilian traffic law, which considers BAC limits equal to zero.

The results of this study show the need for further research,both regarding the exposure of gasoline station attendants toethanol and gasoline, and the effectiveness of the tests performedwith breathalyzers, especially when it comes to traffic laws usingthis test to assess drivers in traffic, also considering the tolerancelimit equal to zero for BAC, such as the Brazilian law.

Acknowledgements

The authors would like to thank CNPq for nancial support andscholarships. Also, the CPAD/HCPA personnel involved in thecollection and analysis of samples, all gasoline station atten-dants who volunteered, all gasoline stations that participated,the State Traffic Department of Rio Grande do Sul (DETRAN-RS)by lending the breathalyzer, and Immunalysis Corporation forthe donation of Quantisal™ OF collection devices, lters, andpreservative buffer solution.

References

1 UNODC, United Nations Office on Drugs and Crime, WorldDrug Report 2013, United Nations Publication, New York,2013.

Anal. Methods

2 WHO, Global Status Report on Road Safety 2013, WorldHealth Organization, Geneva, 2013.

3 H. Gjerde, T. R. Sousa, R. De Boni, A. S. Christophersen,R. P. Limberger, I. Zancanaro, E. L. Øiestad,P. T. Normann, J. Mørland and F. Pechansky, Acomparison of alcohol and drug use by random motorvehicle drivers in Brazil and Norway, Int. J. Drug Policy,2014, 25, 393.

4 H. Gjerde, A. S. Christophersen, P. T. Normann andJ. Mørland, Associations between substance use among carand van drivers in Norway and fatal injury in road trafficaccidents: a case-control study, Transport. Res. F: TrafficPsychol. Behav., 2013, 17, 134.

5 Brasil, Conselho Nacional de Transito, Resoluçao no. 432, de23 de janeiro de 2013, Diario Ocial da Uniao, Brasılia, 2013.

6 M. Passagli and P. A. Marinho, in Toxicologia Forense: Teoriae Pratica, ed. M. Passagli, Millennium, Campinas, 2011.

7 S. H. M. Rabello, M.Sc. dissertation, Universidade FederalFluminense, 2004.

8 E. M. Caravati and K. T. Anderson, Breath Alcohol AnalyzerMistakes Methanol Poisoning for Alcohol Intoxication, Ann.Emerg. Med., 2010, 55, 198.

9 N. J. P. Paulisson and F. Winquist, Analysis of breath alcoholwith a multisensor array: instrumental setup,characterization and evaluation, Forensic Sci. Int., 1999,105, 95.

10 T. J. Buckley, D. P. Joachim, J. R. Bowyer and J. M. Davis,Evaluation of methyl tert-butyl ether (MTBE) as aninterference on commercial breath-alcohol analyzers,Forensic Sci. Int., 2001, 123, 111.

11 E. Spinelli, Vigilancia Toxicologica: comprovaçao do uso dealcool e drogas atraves de testes toxicologicos, Interciencia,Rio de Janeiro, 1st edn, 2004.

12 E. Bernal, in Gas Chromatography in Plant Science, WineTechnology, Toxicology and some Specic Applications, ed. B.Salih and O. Çelikbıçak, InTech, Mexico, 2012.

13 I. A. Was, A. H. Al-Awadhi, Z. N. Al-Hatali and F. J. Al-Rayami, Rapid and sensitive static headspace gaschromatography/mass spectrometry method for theanalysis of ethanol and abused inhalants in blood, J.Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2004, 799,331.

14 Champman and Hall, Dictionary of Organic Compounds,Champaman, New York, London, Toronto, 1982.

15 S. Scivoletto, A. Malbergier and A. Pileggi, in Fundamentos detoxicologia, ed. S. Oga, Atheneu, Sao Paulo, 2008, pp. 389–403.

16 W. Gubala and D. Zuba, Gender differences in thepharmacokinetics of ethanol in saliva and blood aer oralingestion, Pol. J. Pharmacol., 2003, 55, 639.

17 E. E. Elamin, A. A. Masclee, J. Dekker and D. M. Jonkers,Ethanol metabolism and its effects on the intestinalepithelial barrier, Nutr. Rev., 2013, 71, 483.

18 J. M. F. Vieira, Ph.D. thesis, Universidade Fernando Pessoa,2012.

19 A. I. Cederbaum, Alcohol Metabolism. Clin. Liver Dis., 2012,16, 667.

This journal is © The Royal Society of Chemistry 2015

Paper Analytical Methods

Publ

ishe

d on

24

Febr

uary

201

5. D

ownl

oade

d by

Uni

vers

idad

e Fe

dera

l do

Rio

Gra

nde

do S

ul o

n 18

/03/

2015

11:

09:3

4.

View Article Online

20 W. Gubala and D. Zuba, Saliva as an alternative specimen foralcohol determination in the human body, Pol. J. Pharmacol.,2002, 54, 161.

21 E. J. Cone andM. A. Huestis, Interpretation of oral uid testsfor drugs of abuse, Ann. N. Y. Acad. Sci., 2007, 1098, 51.

22 O. H. Drummer, D. Gerostamoulos, M. Chu, P. Swann,M. Boorman and I. Cairns, Drugs in oral uid in randomlyselected drivers, Forensic Sci. Int., 2007, 170, 105.

23 W. M. Bosker and M. A. Huestis, Oral uid testing for drugsof abuse, Clin. Chem., 2009, 55, 1910.

24 H. Gjerde, P. T. Normann, A. S. Christophersen,S. O. Samuelsen and J. Mørland, Alcohol, psychoactivedrugs and fatal road traffic accidents in Norway: a case-control study, Accid. Anal. Prev., 2011, 43, 1197.

25 M. Chu, D. Gerostamoulos, J. Beyer, L. Rodda, M. Boormanand O. H. Drummer, The incidence of drugs of impairmentin oral uid from random roadside testing, Forensic Sci. Int.,2012, 215, 28.

26 I. Zancanaro, R. P. Limberger, P. O. Bohel, M. K. Santos,R. B. De Boni, F. Pechansky and E. D. Caldas, Prescriptionand illicit psychoactive drugs in oral uid-LC-MS/MSmethod development and analysis of samples fromBrazilian drivers, Forensic Sci. Int., 2012, 223, 208.

27 D. Z. Souza, P. O. Bohel, E. Comiran, K. C. Mariotti,F. Pechansky, P. C. A. V. Duarte, R. B. De Boni,P. E. Froehlich and R. P. Limberger, Determination ofamphetamine-type stimulants in oral uid by solid-phasemicroextraction and gas chromatography/massspectrometry, Anal. Chim. Acta, 2011, 696, 67.

28 K. Langel, H. Gjerde, D. Favretto, P. Lillsunde, E. L. Øiestad,S. D. Ferrara and A. G. Verstraete, Comparison of drugconcentrations between whole blood and oral uid, DrugTest. Anal., 2014, 6, 461.

29 M. Yonamine, Ph.D. thesis, Universidade de Sao Paulo, 2004.30 E. Comiran, D. Z. Souza, P. O. Boehl, K. C. Mariotti,

F. Pechansky, P. C. A. V. Duarte, R. B. De Boni,P. E. Froehlich and R. P. Limberger, Fenproporex andAmphetamine Pharmacokinetics in Oral Fluid AerControlled Oral Administration of Fenproporex, Ther. DrugMonit., 2012, 5, 545.

31 N. Malathi, S. Mythili and H. R. Vasanthi, SalivaryDiagnostics: a Brief Review, I.S.R.N. Dentistry, 2014, vol.2014, p. 1.

32 H. Gjerde and A. Verstraete, Can the prevalence of highblood drug concentrations in a population be estimated byanalysing oral uid? a study of tetrahydrocannabinol andamphetamine, Forensic Sci. Int., 2010, 195, 153.

33 E. J. Cone, Legal, workplace, and treatment drug testing withalternate biological matrices on a global scale, Forensic Sci.Int., 2001, 121, 7.

This journal is © The Royal Society of Chemistry 2015

34 Y. H. Caplan and B. A. Goldberger, Alternative specimens forworkplace drug testing, J. Anal. Toxicol., 2001, 25, 396.

35 R. E. Choo and M. A. Huestis, Oral uid as a diagnostic tool,Clin. Chem. Lab. Med., 2004, 42, 1273.

36 L. H. P. Bueno, R. H. A. Da Silva, A. V. Azenha, M. C. S. Diasand B. S. De Martinis, Oral uid as an alternative matrix todetermine etanol for forensic purposes, Forensic Sci. Int.,2014, 242, 117.

37 A. W. Jones, Pharmacokinetics of ethanol in saliva:comparison with blood and breath alcohol proles,subjective feelings of intoxication and diminishedperformance, Clin. Chem., 1993, 39, 1837.

38 M. K. Santos, B. T. Borille, G. N. F. Cruz, B. C. Coppe,E. Comiran, S. Kaiser, P. E. Froehlich and R. P. Limberger,Extraction optimization using Box–Behnken design andmethod validation for ethanol in oral uid, Anal. Methods,2014, 6, 6095.

39 J. Rossy and J. Vasconcelos, in Aperfeiçoamento em tecnicaspara scalizaçao do uso de alcool e outras drogas no transitobrasileiro, ed. F. Pechansky, L. Von Diemen and V. M.Gonçalves, SENAD, Brasılia, 2014.

40 A. Nery-Filho, M. G. Medina, A. G. Melcop and E. M. Oliveira,Impacto do uso do alcool e outras drogas em vıtimas deacidentes de transito, Associaçao Brasileira dosDepartamentos Estaduais de Transito, Brasılia, 1997.

41 C. G. Carvalho, B. C. Contrim, O. A. Silva and N. Sauaia,Blood alcohol content prevalence among trauma patientsseen at a level 1 trauma center, Annu. Rev. Public Health,2002, 36, 47.

42 P. Mura, P. Kintz, B. Ludes, J. M. Gaulier, P. Marquet,S. Martin-Dupont, F. Vincent, A. Kaddour, J. P. Goulle,J. Nouveau, M. Moulsma, S. Tilhet-Coartet and O. Pourrat,Comparison of the prevalence of alcohol, cannabis andother drugs between 900 injured drivers and 900 controlsubjects: results of a French collaborative study, ForensicSci. Int., 2003, 133, 79.

43 R. E. Pereira, S. Perdona Gda, L. C. Zini, M. B. Cury,M. A. Ruzzene, C. C. Martin and B. S. De Martinis,Relation between alcohol consumption and trafficviolations and accidents in the region of Ribeirao Preto,Sao Paulo State, Forensic Sci. Int., 2011, 207, 164.

44 W. H. Schneider, P. T. Savolainen, D. Van Boxel andR. Beverley, Examination of factors determining fault intwo-vehicle motorcycle crashes, Accid. Anal. Prev., 2012, 45,669.

45 A. W. Jones, Evidence-based survey of the elimination ratesof ethanol from blood with applications in forensiccasework, Forensic Sci. Int., 2010, 200, 1.

Anal. Methods